catalyzing CO2 reduction activity. Recently, a
novel, hydrogen-dependent CO2 reductase, purified from the model acetogenic organism Acetobacterium woodii, was identified that fixes CO2
to formate via the electron shuttle H2 ( 68, 69).
Furthermore, the ability for enzymes such as
formate dehydrogenase to catalyze the reduction of CO2 has also been discovered ( 70), and
further characterization and discovery can be exploited to better understand and improve CO2
utilization processes. Of special interest is the
form of the electron donor, which can potentially
be coupled to other biological reactions to maximize overall efficiency.

What is the future of
industrial biomanufacturing?

Despite the promise of C1 feedstocks for industrial biomanufacturing of fuels and chemicals,
much work remains to develop biocatalysts exhibiting the required overall carbon and energy
conversion efficiencies. Biological conversion rates
are currently lower than chemical conversion
rates, and when considered alongside the challenges of potential product toxicity and a dilute
product output due to high water content, additional robust bioprocess development is required
to reach metrics amenable for commercial scale
operation. This will require improvement in both
biocatalyst design and process development and
will rely on continued advancement in a number
of fields, including metabolic engineering and
synthetic and systems biology, among others.
These advancements are critical for the development of bioconversion processes that can exploit the opportunity presented by currently
wasted, distributed methane feedstocks. Although
more mature bioconversion technologies targeting other feedstocks are currently available, no
other feedstock is as widespread and currently
available as methane. This further underscores
the ability to benefit from the learning-by-doing
approach to drive down CapEx costs and advance industrial biomanufacturing as a whole.
Given this potential, reducing time required for
biocatalyst and process development is essential
for industrial manufacturing to capitalize on this
current opportunity and advance the future of
the industry.

Strategies and tools within the iterative de-sign, build, test, and learn cycle of biocatalystdevelopment remain a critical factor for unlock-ing the potential of new approaches to biocon-version. Expanding available metabolic pathwaysthrough the development of novel synthetic en-zymes and pathways to improve the carbon andenergy efficiency of conversion is critical to bothsupport and improve native pathways and orga-nisms engineered for product synthesis. Aidingthese efforts are the use of in silico organism de-sign strategies ( 71), genome mining techniques( 72), and computational enzyme design efforts( 73), in addition to exploiting emerging areassuch as substrate channeling ( 74). As opposed tothe traditional design of single microorganismsfor desired function, the use of microbial con-sortia is an additional emerging approach thatcan potentially target applications challengingfor a single engineered organism ( 75). Theseinclude engineering naturally occurring micro-bial consortia while also developing syntheticconsortia to improve efficiency, stability, andcontrol. Beyond the traditional single-cell bio-catalyst, another approach uses synthetic cell-free systems, which convert a substrate to aproduct using a mixture of enzymes and co-factors for the cascade of reactions ( 76, 77). Thesein vitro systems provide the potential for greatercontrol and flexibility by uncoupling productsynthesis from cellular viability, complexity, andphysiology and enabling high catalyst loadingto support high product titer and productivity.

Advancements in material design and three-dimensional printing have the potential to improve these efforts, with the use of printable
particulate methane monooxygenase-embedded
materials for conversion of methane to methanol
demonstrating the potential to improve not only
biocatalysts but also bioreactor and process
design ( 78).

Genome engineering technologies for making
mutations to microbial genes and genomes in a
targeted, multiplexed manner, such as CRISPR/
Cas9-based systems ( 79–81) and multiplexed automated genome engineering ( 82), are becoming
standard tools for model industrial organisms.
The use of these tools allows in vivo construction
of the millions of potential biosynthetic pathway
variants generated by in silico metabolic methods and design in a fraction of the traditional
time. Furthermore, continued advances in synthetic biology aimed at the development of defined and standardized biological parts can be
exploited for the construction of minimal synthetic genomes and organisms with desired
function ( 83). This type of modularization using
standard parts can improve the automation of
construction, serving to reduce both the time
and the costs associated with biocatalyst development and improve upstream process design
efficiency.

The increasing throughput of design and construction requires increasingly robust methods
for testing cellular functions that go beyond the
traditional analytical tools used today. Screening
and selection tools based on biosensors can help
increase throughput and decrease analysis time
( 84). These techniques can be integrated with an
evolutionary approach to strain development,
with systems biology approaches enabling the
discovery of the genetic and physiological basis
of evolved phenotypes ( 85). Systems biology
tools, such as next-generation sequencing, high-sensitivity proteomic and metabolomic methods,
and developments in fluxomic techniques, can
also be used to analyze and guide additional
rounds of rational design for further optimization and biocatalyst performance refinement,
including improving the tolerance of biocatalysts to desired designer products ( 86).

Even with advancements in biocatalyst devel-opment, a critical challenge is scaling productionfrom typical laboratory conditions used for straindevelopment to those required on a commercialscale without the loss of performance. Typicalbioprocess development takes 5 to 10 years andcan be considerably more expensive than chem-ical technologies owing to the relative infancy ofthe process ( 87). The holistic consideration ofprocess parameters, including product purityand waste-stream management, at an early stageis desirable to develop small-scale testing pro-tocols mimicking required larger-scale processparameters. Modeling and designing novel bio-reactors, especially those for gas-intensive fer-mentations, will be critical to improving thereproducibility and predictability of bioconver-sion processes at the commercial scale. Processintensification can build on initial success incommercial-scale gas fermentation, with theflexibility and modularity of biocatalyst devel-opment enabling direct integration within theprocess to shift product output. Early-stage as-sessment of fermentation product separationis also critical to success, because defining therequirements for separation from a product, pro-cess, and cost standpoint can help guide biocatalystdevelopment efforts and ensure full process in-tegration. All of these process elements will ben-efit from technological learning as bioconversionprocesses are continually explored and devel-oped, which will serve to reduce production andenergy costs and drive process optimization andintegration.

Targeting distributed methane feedstocks currently inaccessible through chemical processes
can help solidify industrial biomanufacturing
as an alternative to industrial chemical manufacturing and serve as an enabling factor for continued expansion and adoption for targeting a
range of complex feedstocks and products. Although much work remains, the strengths and
diversity of biology can be leveraged to further
develop bioconversion processes for rapid, mobile, and widespread deployment outside of those
previously demonstrated. This continued expansion can further facilitate the ability for bioconversion to quickly adapt to market changes
or the arrival of new markets. Together, this
provides clear advantages for addressing the
emerging environmental, geographical, political, and economic factors for industrial chemical manufacturing. In addition to contributing
to solving complex challenges facing our planet,
the smaller scale and lower process complexity of
bioconversion processes also represent a promising and potentially transformative approach to
meet the demands of deep space exploration.
Extraterrestrial in situ resource use, product
manufacturing, and human health demands,
among others, will require innovative production of manufacturing necessities using available
natural resources on location, such as atmospheric gases ( 88, 89). Addressing the issue of
in situ resource use through biomanufacturing
solutions, given the potential for small-scale,
CapEx-efficient design, relative product flexibility, and mild operating conditions will use
an innovative commercial technology in a novel
setting for potentially transformative solutions.
All told, the future of industrial biomanufacturing
Clomburg et al., Science 2017 355, eaag0804 6 January 2017 8 of 10